|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
Originally published as JCO Early Release 10.1200/JCO.2005.01.128 on December 21 2004 © 2005 American Society of Clinical Oncology. Autoimmunity in a Phase I Trial of a Fully Human Anti-Cytotoxic T-Lymphocyte Antigen-4 Monoclonal Antibody With Multiple Melanoma Peptides and Montanide ISA 51 for Patients With Resected Stages III and IV MelanomaFrom the Departments of Medicine, Urology, and Preventive Medicine, Keck/University of Southern California School of Medicine, Los Angeles; Department of Medicine, Stanford University School of Medicine, Stanford, CA; and Medarex Inc, Bloomsbury, NJ Address reprint requests to Jeffrey Weber, MD, PhD, Department of Medicine, Keck School of Medicine, University of Southern California/Norris Cancer Center, 1441 Eastlake Ave, Los Angeles, CA 90033; e-mail: jweber{at}usc.edu
PURPOSE: Nineteen patients with high-risk resected stage III and IV melanoma were immunized with three tumor antigen epitope peptides from gp100, MART-1, and tyrosinase emulsified with adjuvant Montanide ISA 51 and received a fully human anti-cytotoxic T-lymphocyte antigen-4 (anti–CTLA-4) monoclonal antibody MDX-010. Each of three cohorts received escalating doses of antibody with vaccine primarily to evaluate the toxicities and maximum-tolerated dose (MTD) of MDX-010 with vaccine. MDX-010 pharmacokinetics and immune responses were secondary end points. PATIENTS AND METHODS: Peptide immunizations with MDX-010 were administered every 4 weeks for 6 months and then every 12 weeks for 6 months. A leukapheresis to obtain peripheral-blood mononuclear cells for immune analyses was performed before treatment and after the sixth vaccination. Patients were observed until relapse. RESULTS: Grade 3 gastrointestinal (GI) toxicity (diarrhea or abdominal pain) was observed in three patients in the highest dose cohort and one in the middle dose cohort who seemed to be autoimmune. That defined the MTD with vaccine on this schedule at 1 mg/kg. Of eight patients with evidence of autoimmunity, three have experienced disease relapse. Of 11 patients without autoimmune symptoms, nine have experienced disease relapse. Significant immune responses were measured by tetramer and enzyme-linked immunospot assays against gp100 and MART-1. CONCLUSION: Dose-related autoimmune adverse events, predominantly skin and GI toxicities, were reversible. Patients mounted an antigen-specific immune response to a peptide vaccine when combined with a human anti–CTLA-4 antibody.
T-cell activation and expansion after immune stimuli lead to contraction and generation of central memory cells. This maintains immune homeostasis and downregulates responses to self while targeting nonself antigens.1,2 Cytotoxic T-lymphocyte (CTL) antigen-4 (CTLA-4) is thought to be of primary importance in maintaining immune homeostasis and the induction of tolerance to self antigens.3-5 It mediates its effects through inhibitory T-cell signaling and blockade of the CD28-B7 costimulatory pathway.3-8 CTLA-4 knockout mice develop a lymphoproliferative syndrome with multiorgan lymphocytic infiltration and tissue destruction, and die by 3 to 4 weeks.9 CTLA-4–related tolerance induction is T-cell dependent because depletion of CD4 T cells from birth prevents lymphoproliferation in CTLA-4–deficient mice10; the same mice bred with Rag–/– mice devoid of T cells do not develop the disease. CTLA-4 terminates T-cell expansion and arrests T cells in G1 of the cell cycle.11,12 Interferon gamma secretion from activated T cells is diminished in the presence of CTLA-4 stimulation.13 Inhibition of CTLA-4 activity with anti–CTLA-4 antibody allows T-cell expansion to continue after vaccination with tumor antigen.14 Overcoming tolerance may be important in a successful cancer vaccine strategy. Given that many human tumor-associated antigens (TAAs) are unmutated self antigens, an effective response against many neoplasms requires targeting immunity to self antigens and overcoming tolerance. To date, strategies aimed at producing a CTL response toward TAAs have rarely resulted in tumor regression. Manipulation of CTLA-4 holds promise as an anticancer therapy because of its pivotal role in T-cell–dependent tolerance. Numerous experimental models have demonstrated the ability of CTLA-4 blockade to break tolerance in vivo as indicated by increased levels of autoimmunity and improved responses to tumor vaccines.15,16 To test whether abrogation of CTLA-4 signaling in patients receiving a melanoma vaccine broke tolerance and induced a response to self antigens, we tested repeated dosing of a human anti–CTLA-4 monoclonal antibody (MDX-010) administered with a multipeptide vaccine with adjuvant Montanide ISA 51 to high-risk patients with resected stages III and IV melanoma. Principal end points were the toxicities and maximum-tolerated dose (MTD) of the vaccine with MDX-010, and secondary end points included immune responses against melanoma antigens and pharmacokinetics of MDX-010. MDX-010 induced dose-dependent and reversible autoimmune adverse effects that may correlate with the risk of relapse, suggesting that development of autoimmunity might correlate with clinical benefit and antimelanoma immunity.
Patients had resected stage III or IV melanoma by the modified American Joint Commission on Cancer staging system (2001) and were rendered free of disease surgically. They had a magnetic resonance imaging or computed tomographic scan of the brain and computed tomographic imaging of the chest, abdomen, and pelvis performed within 4 weeks of therapy showing no evidence of disease. Eligibility criteria included age 18 or older, creatinine less than 2.0 mg/dL, bilirubin less than 2.0 mg/dL, platelet count 100,000/µL or more, hemoglobin of 9 g/dL or more, and total WBC of 3000/µL or more. HIV, hepatitis C antibody, and hepatitis B surface antigen titers were required to be negative. All patients were HLA-A*0201-positive by a DNA polymerase chain reaction assay. Patients were required to comprehend and sign an informed consent form approved by the Cancer Therapy Research Program of the National Cancer Institute, and the Los Angeles County and University of Southern California institutional review boards. Exclusion criteria included active autoimmune disease, corticosteroid dependence, and prior treatment with MDX-010 antibody or MART-1, gp100, and tyrosinase peptides. Primary end points of the trial were the adverse effects and tolerability of MDX-010, and the MTD of MDX-010 with a vaccine. Secondary end points included pharmacokinetics of MDX-010 and immunologic responses to vaccine with MDX-010. MDX-010 (provided by Medarex Inc, Bloomsbury, NJ) was administered intravenously during 90 minutes every 4 weeks for 6 months, then every 12 weeks for 6 months. Antibody infusions were accompanied by three separate subcutaneous injections of 1 mg of each peptide emulsified in Montanide ISA 51. Patients received MDX-010 at 0.3, 1.0, or 3.0 mg/kg, followed by subcutaneous injection of tyrosinase368-376 (370D), gp100209-217 (210M), and MART-126-35 (27L) peptides. Leukapheresis with exchange of 5 to 7 L for peripheral-blood mononuclear cells (PBMCs) was performed immediately before and 6 months after the initial vaccination. PBMCs were frozen at –168°C as previously described.17 Patients were observed until relapse. Tetramers were produced in accordance with the approach of Altman.18 The tetramer assay technique was previously published by our group,19,20 and was validated by staining against a CTL line or clone specific for HLA-A2 in association with the peptide of interest. The limit of detection was 0.01% of CD8+ T cells as described.20 Thawed PBMC rested overnight and were tested in an enzyme-linked immunospot assay (ELISPOT) as previously described.20 PBMCs were added at 166,000, 83,000, and 41,500 cells per well in triplicate in a volume of 100 µL to membrane plates (MAHA S45-10; Millipore, Bedford, MA), washed, and prepared with primary anti-interferon gamma antibody (MabTech, Nacka, Sweden). Phytohemagglutinin at 10 µg/mL was added to six wells as a positive control, and AIM-V (GIBCO Inc, Grand Island, NY) media was added as a negative control. Peptides were added at 5 µg/mL to all other wells. Plates were incubated for 4 hours at 37°C. Secondary antibody (MabTech) at 1 mg/mL was added to plates, which were incubated overnight at 4°C. Plates were developed in the dark, and the colorimetric reaction was halted by washing with running water. Plates were read on a KS Elispot reader (Carl Zeiss, Thornwood, NY) and normalized to spots per 100,000 CD8 T cells. Negative controls included the human papillomavirus E7 86 to 93 A*0201 restricted peptide, for which geometric mean values were characteristically less than 10 spots per 105. The tyrosinase368-376 (370D) peptide (NSC No. 699048), MART-126-35 (27L) peptide (NSC No. 709401), and gp100209-217 (210M) peptide (NSC No. 683472) HLA-A*0201 restricted nine or 10 amino acid epitope peptides and were prepared to good manufacturing practices grade and administered as described.20 They were produced by Ben Venue Laboratories Inc (Bedford, OH). Peptide was provided by the Cancer Therapy Evaluation Program of the National Cancer Institute (Bethesda, MD) under an Investigational New Drug application BB 6123. Montanide ISA-51 (incomplete Freund's adjuvant [IFA]) was manufactured by Seppic Inc (Franklin Lakes, NJ) and supplied by Cancer Therapy Evaluation Program of the National Cancer Institute in glass ampules containing 3 mL of sterile adjuvant solution without preservative. Fully human MDX-010 (anti–CTLA-4 monoclonal antibody; immunoglobulin G1 [IgG1] antibody) was supplied at a concentration of 5 mg/mL in vials containing 5 or 10 mL, and was stored between 2°C and 8°C. MDX-010 was drawn through a 0.22 µmol/L filter, and diluted in normal saline to a concentration of 2.5 mg/mL. Skin tests were performed as previously described.17 All patients had a complete skin examination before therapy and at each vaccination visit to screen for vitiligo. Ocular toxicity was determined as previously described.20 PBMCs were stained with fluorescein isothiocyanate–labeled anti-CD8, anti–chemokine receptor 9, anti–common leukocyte antigen, and phycoerythrin-conjugated peptide/HLA-A2.1 tetramers, as well as CCR4 antibodies, and Cy5-labeled anti-CD4, anti-CD14, and anti-CD19 antibodies at 4°C for 30 minutes. Cells were washed and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) as described.20 Immunohistochemical staining of paraffin-imbedded sections on glass slides for HMB-45, MART-1, and tyrosinase was performed as described.20 Appropriate negative and positive control sections were included with each assay. Archival formalin-fixed, paraffin-embedded tissue was sectioned at 5-µm intervals and mounted on charged slides (ProbeOn+, Fisher Scientific, Pittsburgh, PA). Sections were deparaffinized and rehydrated through graded alcohols, then subjected to antigen retrieval. The tissue was incubated with a blocking serum (usually 5% horse serum); the blocking serum was aspirated and the primary monoclonal antibody applied in the appropriate dilution. Anti-CD4 and anti-CD8 monoclonal antibodies (Becton Dickinson, Mountain View, CA) were used for immunohistochemical staining. In all cases, external and internal controls were used to assess the quality of the immunohistochemistry reaction. Genomic DNA was isolated from PBMCs using a QiaAmp kit (Qiagen, Valencia, CA). Genotypes for CTLA-4 (JO33) were analyzed using polymerase chain reaction–restriction fragment length polymorphism techniques, as described previously.21 Quantitative, functional enzyme-linked immunosorbent assay was used to determine plasma levels of MDX-010. Plasma samples stored at –80°C were thawed, and then incubated on a plate coated with recombinant human CTLA-4/Fc chimeric protein. Bound MDX-010 was detected with an antihuman IgG, F(ab')2 alkaline phosphatase probe. A standard curve was generated and plasma levels were calculated from the linear portion of the standard curve. Semiquantitative enzyme-linked immunosorbent assay was used to determine the level of plasma anti-MDX-010 IgG. Plates were coated with MDX-010 F(ab')2 and antibodies were detected with an antihuman IgG, Fc-specific conjugated probe. The data were expressed as the inverse of the highest dilution of the plasma sample that generated a background measured at 0.100 optical density. The results for each sample were expressed as fold increase in titer relative to a pretreatment sample. Samples with greater than four-fold increases were considered to be positive. For the tetramer assays, for each peptide, the difference between pretreatment and post-treatment levels was calculated; the signed rank test was used to test for net change after treatment. ELISPOT studies were done in triplicate. Means for each patient for each peptide were calculated pre- and post-treatment. For each patient and peptide, the difference between post-treatment and pretreatment mean was calculated; the signed rank test was used to compare the post-treatment mean number of spots to the pretreatment mean number of spots. CCR9 expression analysis was performed in duplicate for some patients and once in others; for this immune measure, the log transformation was taken before analysis and means were calculated in the log scale. Using the post-treatment –pretreatment difference, the weighted t test was used to test for change in CCR9 expression after vaccination. All reported P values are two-sided.
Demographics Nineteen patients were treated. Seven had resected stage IV disease, and the remaining 12 had resected stage III disease (Table 1). Eight received prior immunotherapy with a cell vaccine or interferon alfa-2a, two received biochemotherapy, and all had undergone surgery. Median time from primary diagnosis was 20 months. Seven patients received MDX-010 at 0.3 mg/kg per dose (cohort 1), seven received 1.0 mg/kg (cohort 2), and five received 3.0 mg/kg (cohort 3). Because of US Food and Drug Administration requirements, the first seven patients were dosed every 4 weeks with MDX-010 if their serum antibody level was  2 µg/mL 1 week before the scheduled infusion. This requirement was lifted after patient eight in cohort two.
Autoimmune Toxicity Toxicities including local pain, swelling, granuloma formation, fevers, and flu-like symptoms occurred in the majority of patients, principally of grades 1 and 2 (Table 2). The extraordinary toxicities observed were uveitis and gastrointestinal (GI) toxicity in one patient and a rash in two other patients from cohort 2, and GI toxicity in all five patients from cohort 3. Three patients from cohort 3 experienced grade 2 diarrhea and the remaining two patients developed grade 3 diarrhea. The patient from cohort 2 who developed uveitis and grade 3 bloody diarrhea required hospitalization. One patient from cohort 3 required admission for severe grade 3 abdominal pain and grade 2 diarrhea necessitating intravenous opiates and supportive management. Eight patients had evidence of toxicities believed to be related to MDX-010 that may have been autoimmune in etiology. Three of five patients in the 3 mg/kg cohort experienced dose-limiting toxicity, ending the trial at 19 patients and defining the MTD at 1 mg/kg.
Patient 8 in cohort 2 developed a grade 2 rash on her left breast after the second MDX-010 injection. A biopsy revealed a deep dermal infiltrate of CD4+ T cells with thickened dermis and perivascular cuffing, without true vasculitis (Fig 1A). A lesser infiltrate with CD8+ T cells was also seen (not shown). The patient's vaccination regimen was not interrupted. The rash resolved slowly during 6 months.
Patient 19 in cohort 2 developed self-limited grade 2 diarrhea after the first MDX-010 infusion. One week after his second infusion, the patient presented with bloody diarrhea, visual impairment, photophobia, fevers, and fatigue. Colonoscopy revealed diffuse inflammation of the rectum without ulceration. The rectal biopsy obtained during the colonoscopy revealed a predominant CD4+ T-cell infiltrate sparing the glands (Fig 1B). A lesser infiltrate with CD8+ T cells was also seen in the rectal interstitium (not shown). This was the only patient to require corticosteroids for the treatment of uveitis and colitis, with resolution of all symptoms within 60 days.
Laboratory Values Flow cytometry analyses were performed on PBMC specimens at the time of the first, second, and third MDX-010 infusions, as described above. CD3+, CD4+, CD8+ T cells, CD56+ NK cells, and CD19+ B cells were enumerated. There were no consistent changes over time in those subsets. Activation markers CD69 and HLA-DR, as well as regulatory marker CD25, were also measured on T cells. The only significant change noted was an increase over time in CD4+/HLA-DR+ T cells at the 1 and 3 mg/kg doses, reflective of an increased population of activated T-helper cells (data not shown).
Autoimmune Parameters
Pharmacokinetic Assays
Immune Responses Seventeen of 19 patients completed leukapheresis before and 6 months after vaccination with peptides plus IFA and MDX-010. Immune responses to MART-1, gp100, and tyrosinase were measured using ELISPOT and tetramer assays. The functional ELISPOT assay measures antigen-specific activation of CD8+ T cells secreting interferon gamma. Seventeen patients were evaluated, showing that 15 of 17 had 10+ spots per 105 effectors (an average based on three replicates) to the gp100209-217 (210M) peptide (Fig 3A), and 16 of 17 had 10+ spots in response to the MART-126-35 (27L) peptide (Fig 3B). On average, the number of spots increased by 70% (P = .049) to the gp100 peptide and 24% to MART peptide (P = .096). On the basis of the defined criteria for ELISPOT response, six patients (35%) had a response to gp100 and five patients had a response to MART-1 (29%); eight patients (47%) had a response to one or the other peptide. High levels of prevaccine reactivity to MART-1 were noted in four patients, whereas prevaccine reactivity to gp100 was generally low. There was no difference in ELISPOT reactivity with vaccine dose or relation to autoimmune toxicity, although the limited number of patients precludes making definitive correlations. A similar proportion of patients had an immune response to the wild-type gp100209-217 (six of 17) and MART-127-35 peptides (three of 17), but with a lower amplitude of response (data not shown). No significant ELISPOT reactivity to tyrosinase was observed.
The tetramer assay enumerates CD8+ T cells specific for a class I peptide combination. The results of gp100209-217 (210M) tetramer assays pre- and postvaccine at 6 months after six vaccinations are shown in Fig 4A. No pre-existing reactivity to gp100 was observed, but after vaccination 11 of 16 (68%) patients had a significant increase in the number of gp100209-217 (210M)–specific T cells (median response of 0.042% CD8+ T cells; P < .001). In Fig 4B, six of 16 (37%) patients (who were all negative initially) demonstrated a MART-1 response (median increase in the percent of CD8+ T cells of 0.052%; P = .073) after vaccination. Strong pre-existing reactivity to MART-1 is demonstrated clearly for seven patients, none of whom met the criteria for a response. No significant tetramer reactivity to tyrosinase was observed. A similar but lower proportion of patients had evidence of reactivity to the gp100209-217 (seven of 16) and MART-127-35 (five of 16) wild-type peptides, but a lower level of reactivity was observed (data not shown). There was no difference in tetramer reactivity with vaccine dose or relation to autoimmune toxicity. Only tetramer reactivity to gp100 correlated with time to relapse (P < .001).
Polymorphisms At least four single nucleotide polymorphisms for CTLA-4 have been identified. One of these, JO33 encodes three alleles that correlate with the level of CTLA-4 expression on T cells. The GG allele for that polymorphism correlated with low CTLA-4 levels was shown to be associated with juvenile-onset diabetes and other autoimmune disorders.22 We hypothesized that patients with low CTLA-4 alleles (GG) would have a higher chance of developing autoimmunity with CTLA-4 blockade, and that high CTLA-4 alleles (AA or AG) would have a lesser effect from MDX-010 blockade and a lower chance of developing autoimmunity. In fact, three of four (75%) patients with the low CTLA-4 allele (GG) developed autoimmune symptoms and only two of these four (50%) patients has experienced disease relapse. All four patients received the 1 or 3 mg/kg dose of MDX-010. Of the remaining 15 patients with either the AA or AG alleles, only five (33%) developed autoimmune symptoms and 10 (67%) have experienced disease relapse (Fig 5). This intriguing result is consistent with previous findings that implicate CTLA-4 polymorphisms and their effects on CTLA-4 expression and activity in the modulation of autoimmunity. In this preliminary investigation, the GG allele (low CTLA-4 activity) might correlate with improved prognosis in high-risk melanoma patients treated with vaccine plus CTLA-4 blockade, and merits confirmation in a larger cohort of patients treated with MDX-010.
Flow Cytometry for CCR9 Expression Expression of T-cell homing receptors for skin (CLA and CCR4) and GI mucosa (CCR9) might account for cutaneous and GI manifestations observed in patients enrolled onto this trial with autoimmune adverse effects. Flow cytometry analyses were performed using whole PBMCs obtained before and 6 months after starting vaccination, and then PBMCs were stained for the above markers. Results for CCR9 are shown in Fig 6 and indicate that on average, expression of CCR9 on CD4+ T cells increased by 41% after MDX-010 plus vaccination (P = .02). Eight of 13 patients analyzed (62%) had significant increases in staining of CD4+ T cells for CCR9. No significant differences were noted between cohorts, and differences were not related to development of autoimmunity, although total numbers were small. No changes were noted for CD4/CLA or CD4/CCR4 staining (data not shown).
Clinical Results With 28.5 months of follow-up, 12 of the 19 patients treated on this trial have experienced disease relapse and three have died. Of 11 patients with no evidence of autoimmunity, nine (82%) have experienced disease relapse and two have died. Median time to relapse was 18.3 months. Eight of 19 patients showed signs of autoimmune symptoms; three have experienced disease relapse (37%) and one has died. All five (100%) patients in the highest dose cohort (3 mg/kg) had evidence of autoimmunity; three (60%) have experienced disease relapse and four are alive (two are alive with disease). Of the 14 patients in the two lower dose cohorts, only three (21%) suffered autoimmune effects; nine (64%) patients have experienced disease relapse and two have died. These results raise the possibility of a correlation between development of autoimmunity and lack of relapse. No consistent evidence was found of serologic autoimmunity, and no patient developed anti-MDX-010 antibody responses.
Preclinical work supporting this trial suggested that blockade of CTLA-4 signaling would augment vaccination.15,16 In mice, a melanoma cell line producing granulocyte-macrophage colony-stimulating factor (GM-CSF) induced preventive but not therapeutic immunity via cross-priming.23 Addition of anti–CTLA-4 antibody caused complete rejection or delay of established tumors.16 Anti–CTLA-4 antibody induced rejection of both mammary and prostate carcinomas with a GM-CSF vaccine in mice,24,25 and therapeutic effects against melanoma and p53 antigens as well as survival were augmented.15,26 Transgenic mouse experiments suggest that CTLA-4 may dampen pathologic immune responses to self proteins while promoting immunity against agents providing a danger signal.27 CTLA-4 blockade caused autoimmunity and reactivity to self antigens. Rejection of B16 melanoma after immunization with a vaccine with anti–CTLA-4 was accompanied by depigmentation and absence of melanocytes in affected skin.28 Vitiligo was not seen after vaccine alone,16 suggesting that peripheral T-cell tolerance was broken. Vitiligo and elimination of tumors were enhanced in the absence of CD4+ T cells,16,29 suggesting that T regulatory cells might regulate responses to self-differentiation antigens.30 Induction of autoimmune adverse events was shown in this trial; although the numbers are small, fewer patients with autoimmune events seem to have experienced a disease relapse. Our findings are consistent with those recently reported by Phan et al31 in which eight of 14 patients with metastatic melanoma experienced significant autoimmunity after receiving MDX-010 at 3 mg/kg with a gp100 peptide vaccine every 3 weeks. Three patients experienced objective cancer responses that have been maintained for more than 15 months. Clinical responders had autoimmune symptoms that were treated with corticosteroids and resolved. In that study, there was no dose escalation, and prolonged dosing was not achieved because responders discontinued therapy due to toxicity. In the current study, we show that MDX-010 at 1 mg/kg every 4 weeks with vaccine is a well-tolerated dose, that CCR9 expression seems to increase with treatment, and that CTLA-4 polymorphisms should be assessed in future studies. In a single-dose trial of MDX-010 at 3 mg/kg in metastatic melanoma, one of 17 patients responded. No dose-limiting toxicities were encountered but subclinical autoimmunity, including transiently elevated antinuclear antibodies titers and clinically insignificant retinal depigmentation were noted.32 The antibody was also given at 3 mg/kg to nine patients, two with ovarian cancer and nine with melanoma, who had previously received a cell vaccine.33 Biopsies of tumors after vaccination revealed necrosis, with four melanoma patients experiencing regression of individual metastases. We treated 19 resected melanoma patients with a poor expected outcome. Two had reversible grade 2 rash in the cohort at 1 mg/kg, but one of seven patients at that dose experienced dose-limiting toxicity described above. In the highest-dose cohort, all five patients had evidence of autoimmune toxicity manifested by diarrhea; two had dose-limiting grade 3 diarrhea and one had dose-limiting grade 3 abdominal pain and grade 2 diarrhea. No patient in that cohort required corticosteroids. All toxicities reversed to normal during 4 to 8 weeks. The development of autoimmune toxicity was dose related, and both in our trial and the trial of Phan et al31 autoimmune adverse effects were likely due to the combination of vaccine with antibody. In a recent trial of antibody alone at 3 mg/kg, significantly fewer patients exhibited autoimmunity,32 and when patients received repeated doses of antibody alone at 3 mg/kg, or single doses of a similar antibody at 3 mg/kg, fewer than 20% of patients developed autoimmune toxicity.22,34 An increase in activated CD4 T cells that were DR+ was observed, and was also seen in the trial of Phan et al.31 No trend in kidney or liver function abnormalities were seen, and no alterations in serologic measures of autoimmunity were seen. A recent study implicated a CTLA-4 promoter polymorphism that altered the expression and/or function of CTLA-4 in the development of diabetes,35 and numerous reports suggested a correlation between CTLA-4 polymorphisms and autoimmunity. Analysis of CTLA-4 polymorphisms in this trial revealed that there was a modest correlation between a polymorphism (JO33) associated with low levels of CTLA-4 and the onset of autoimmunity (three of four patients), compared with the alleles associated with medium to high levels of CTLA-4 and autoimmunity (five of 15 patients). Only two of four in the low CTLA-4 group experienced relapse, versus 10 of 15 in the alleles associated with medium to high levels of CTLA-4. The level of immunity by fresh tetramer and ELISPOT (68% v 47%) seemed to be greater than that in prior trials with peptides and adjuvant,17,19,20 given that immune response was approximately 10% in a recent trial of peptides with GM-CSF and interleukin-12 (Weber et al, unpublished data), and less than 25% in the peptides and IFA arm in a recent Eastern Cooperative Oncology Group trial.36 The small number of patients in the three cohorts precludes definitive statements about dose response. There was a modest but not significant correlation of dose and clinical autoimmunity with immune response as quantified by these assays. The proportion or type of functional T cells, or the trafficking of those effector cells, may have been influenced by abrogation of CTLA-4 signaling. Alternatively, the immune effects of MDX0-010 may be not be T-cell specific and could act via antibody-mediated immunity or may act on other antigen-specific T cells that we did not measure via epitope spreading. The observation that CD4+/DR+ cells were increased in the circulation of our patients and those of Phan et al,31 and the intense CD4 T-cell infiltrate seen in Fig 1, are consistent with altered T-cell trafficking. Such alterations have been found in CD4 T cells from patients with inflammatory bowel disease, which have been implicated in the development of that autoimmune disorder.37 We plan to prospectively test whether development of autoimmunity and changes in CD4 cells after treatment with MDX-010 is a surrogate for clinical response. The ability to break tolerance to target specific self antigens associated with tumors would be of significant clinical benefit.38 Our trial adds insight into the clinical manifestations of CTLA-4 blockade, specifically the incidence of autoimmunity after vaccination and the potential link to disease relapse.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Employment: Geoffrey Nichol, Medarex; Tibor Keler, Medarex; Michael Yellin; Medarex. Consultant/Advisory Role: Jeffrey Weber, Medarex. Stock Ownership: Geoffrey Nichol, Medarex; Tibor Keler, Medarex; Michael Yellin, Medarex. Research Funding: Jeffrey Weber, Medarex. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration form and the Disclosures of Potential Conflicts of Interest section of Information for Contributors found in the front of every issue.
We gratefully acknowledge the dedication and encouragement shown by the staff of the Cancer Therapy Evaluation Program, National Cancer Institute, particularly Howard Streicher, MD, and James Zweibel, MD. We also acknowledge helpful conversations with Steven Rosenberg of the Surgery Branch, National Cancer Institute.
Supported by a grant from the Food and Drug Administration's Orphan Drug Program (FD-001-01) and by a grant from Medarex Corp. Authors' disclosures of potential conflicts of interest are found at the end of this article.
1. van der Merwe PA, Davis SJ: Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol 21:659-684, 2003[CrossRef][Medline] 2. Davis SJ, Ikemizu S, Evans EJ, et al: The nature of molecular recognition by T cells. Nat Immunol 4:217-224, 2003[CrossRef][Medline] 3. Thompson CB, Allison JP: The emerging role of CTLA-4 as an immune attenuator. Immunity 7:445-450, 1997[Medline]
4. Takahashi T, Tagami T, Yamazaki S, et al: Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 192:303-310, 2000 5. Salomon B, Bluestone JA: Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu Rev Immunol 19:225-252, 2001[CrossRef][Medline]
6. Waterhouse P, Pennington JM, Timms E, et al: Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 270:985-988, 1995 7. Perez VL, van Parijs L, Biuckians A, et al: Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411- 417, 1997[CrossRef][Medline] 8. Shrikant P, Khoruts A, Mescher MF: CTLA-4 blockade reverses CD8+ T cell tolerance to tumor by a CD4+ T cell- and IL-2-dependent mechanism. Immunity 11:483-493, 1999[CrossRef][Medline] 9. Tivol EA, Borriello F, Schweiter AN, et al: Loss of CTLA-4 leads to massive lymphoproliferationand fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541-547, 1995[CrossRef][Medline] 10. Bachmann MF, Gallimone A, Jones E, et al: Normal pathogen-specific immune responses mounted by CTLA-4-deficient T cells: A paradigm reconsidered. Eur J Immunol 31:450-458, 2001[CrossRef][Medline]
11. Yu X, et al: The role of B7 costimulation in CD4/CD8 T cell homeostasis. J Immunol 164:3543-3553, 2000
12. Prlic M, et al: Homeostatic expansion occurs independently of costimulatory signals. J Immunol 167:5664-5668, 2001
13. Alegre ML, Sheis H, Thompson CB, et al: Expression and function of CTLA-4 in Th1 and Th2 cells. J Immunol 161:3347-3356, 1998
14. Huang AY, Golumbek P, Ahmadzadeh M, et al: Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science 264:961-965, 1994
15. Davila E, Kennedy R, Celis E: Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG-oligodeoxynucleotide adjuvant, and CTLA-4 blockade. Cancer Res 63:3281-3288, 2003
16. van Elsas A, Sutmuller RP, Hurwitz AA, et al: Elucidating the autoimmune and anti-tumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 (CTLA-4) blockade in combination with a B16 melanoma vaccine: Comparison of prophylaxis and therapy. J Exp Med 194:427-438, 2001
17. Lee P, Wang F, Kuniyoshi J, et al: Effects of interleukin-12 on the immune response to a multi-peptide vaccine for resected metastatic melanoma. J Clin Oncol 19:3836-3847, 2001
18. Altman J, Moss PAH, Goulder P, et al: Phenotypic analysis of antigen specific T lymphocytes. Science 274:94-96, 1996 19. Weber J, Sondak VK, Scotland R, et al: Granulocyte-macrophage-colony-stimulating factor added to a multipeptide vaccine for resected stage II melanoma. Cancer 97:186-200, 2003[CrossRef][Medline] 20. Pullarkat V, Lee PP, Scotland R, et al: A phase I trial of SD-9427 (progenipoietin) with a multipeptide vaccine for resected metastatic melanoma. Clin Cancer Res 4:1301-1312, 2003 21. Perrey C, Turner SJ, Pravica V, et al: ARMS-PCR methodologies to determine IL-10, TNF-alpha, TNF-beta and TGF-beta 1 gene polymorphisms. Transpl Immunol 7:127-128, 1999[CrossRef][Medline] 22. Camacho LM, Ribas A, Glaspy JA, et al: Phase I trial of human CTLA-4 monoclonal antibody CP-675,206 in patients with advanced solid malignancies. Proc Am Soc Clin Oncol 23:164, 2004 (abstr 2505)
23. Dranoff G, Jaffee E, Lazenby A, et al: Vaccination with irradiated tumor cells engineered to secrete GM-CSF stimulates potent, specific, and long lasting anti-tumor immunity. Proc Natl Acad Sci U S A 90:3539-3543, 1993
24. Hurwitz AA, Yu TF, Leach DR, et al: CTLA-4 blockade synergizes with tumor-derived GM-CSF for treatment of an experimental mammary carcinoma. Proc Natl Acad Sci U S A 95:10067-10071, 1998
25. Hurwitz AA, et al: Combination immunotherapy of primary prostate cancer in a transgenic model using CTLA-4 blockade. Cancer Res 60:2444-2448, 2000
26. Espenschied J, Lamont J, Longmate J, et al: CTLA-4 blockade enhances the therapeutic effect of an attenuated poxvirus vaccine targeting p53 in an established murine tumor model. J Immunol 170:3401-3407, 2003
27. Walker LS, Ausubel LJ, Chodos A, et al: CTLA-4 differentially regulates T cell responses to endogenous tissue protein versus exogenous immunogen. J Immunol 169:6202-6209, 2002
28. van Elsas A, Hurwitz AA, Allison JP: Combination immunotherapy of B16 melanoma using anti–CTLA-4 and GM-CSF producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med 190:355-366, 1999 29. Rosenberg SA, White DE: Vitiligo in patients with melanoma: Normal tissue antigens can be targets for cancer immunotherapy. J Immunother 19:81-84, 1996[CrossRef][Medline]
30. Sutmuller RPM, van Duivenvoorde LM, van Elsas A, et al: Synergism of CTLA-4 blockade and depletion of CD25+ regulatory T cells in anti-tumor therapy reveals alternative pathways for suppression of auto-reactive CTL responses. J Exp Med 194:823-832, 2001
31. Phan GQ, Yang JC, Sherry RM, et al: Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci U S A 100:8372-8377, 2003 32. Tchekmedyian S, Glasby J, Korman A, et al: MDX-010 (human anti-CTLA4): A phase I trial in malignant melanoma. Proc Am Soc Clin Oncol 21:223, 2002 (abstr 56)
33. Hodi FS, Mihm MC, Soiffer RJ, et al: Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc Natl Acad Sci U S A 100:4712-4717, 2003 34. Hersh E, Weber J, Powderly J, et al: A phase II randomized multi center study of MDX-010 alone or in combination with dacarbazine (DTIC) in stage IV metastatic malignant melanoma. Proc Am Soc Clin Oncol 23:709, 2004 (abstr 7511) 35. Ueda H, Howson JM, Esposito L, et al: Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423:506-511, 2003[CrossRef][Medline] 36. Kirkwood JM, Lee S, Land S, et al: E1696: Final analysis of the clinical and immunological results of a multi-center ECOG phase II trial of multi-epitope peptide vaccination for stage IV melanoma with MART-1 27-35 , gp100 209-217 and tyrosinase 368-376 (370D) +/- alpha-IFN +/- GM-CSF. Proc Am Soc Clin Oncol 23:707, 2004 (abstr 7502)
37. Papadakis KA, Landers C, Prehn J, et al: CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile. J Immunol 171:159-165, 2003 38. Watanabe N, Gavrieli M, Sedy JR, et al: BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1. Nat Immunol 4:670-679, 2003[CrossRef][Medline] Submitted January 22, 2004; accepted August 27, 2004. Related Editorials
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
INTRODUCTION
